Our long-term goal is to explore the possibility of developing a new line of analgesics that are devoid of the major side effects of opiates, namely tolerance, physical dependence, gastrointestinal immotility, and respiratory depression, in a study of endogenous neuropeptidase inhibitors and their analogs. Recent findings reveal that, like the protein inhibitors for proteinases, endogenous peptide inhibitors control the bioavailability of enkephalins in brain. Arg0- enkephalin is one of such inhibitors. In this proposal, we plan 1) to identify endogenous cerebral inhibitors, 2) to define their inhibitory specificities toward brain- specific and general peptidases, 3) to study the localization of inhibitors and their effect on cerebral enkephalin distribution, correlated with effects on analgesia and morphine withdrawal, and 4) to study the release of enkephalins and of inhibitors in brain slices and brain cell cultures. We will isolate and purify Arg0-enkephalin and related peptides from bovine striatum by FPLC and HPLC. Their chemical structures will be identified by gas-phase peptide microsequencing and by mass spectrometry. Their co- existence with enkephalins will be determined by highly specific immunohistochemistry and quantified by radioimmunoassays in brain enkephalinergic regions and during development. The study of endogenous anti-enkephalinase will enrich our understanding of the modulation of neuropeptides and their pharmacological effects. In addition, we hope to establish the presence of novel neuropeptides and potentially innovative analgesics in the brain.